Development of an automated high-content immuno-fluorescence assay of pSmads quantification: proof-of-concept with drugs inhibiting the BMP/TGFβ pathways

Abstract

AbstractBone morphogenetic proteins (BMPs) and transforming growth factors (TGF-β) are members of the transforming growth factors superfamily, known for their role in several physiological and pathological processes. These factors are known to bindin vivoto BMP and TGF-β receptors respectively, which induces the phosphorylation of the Smad (pSmad) transcription factors. This pathway is generally studied with western blot and luciferase bioluminescence assay, which present some limitations. In our work, we developed and optimized a high-content immunofluorescence assay to study the pSmad pathway on glass as well as on biomaterials by overcoming the technical challenges raised by image acquisition and analysis. Furthermore, with this assay, we present here a proof-of concept for drug testing on glass and on biomimetic films using drug inhibitors of the BMP receptor and of the TGF-β receptors. Altogether, our results open perspectives for future drug testing on biomimetic films that present various growth factors and extracellular matrix proteins or peptides.